KazukiIde1,HiroshiYamada1*,KumiMatsushita2,MikiIto1,KeiNojiri1,KiichiroToyoizumi1,KeijiMatsumoto1,YoichiSameshima31DepartmentofDrugEvaluationandInformatics,GraduateSchoolofPharmaceuticalSciences,UniversityofShizuoka,Shizuoka,Japan,2DepartmentofPharmacy,KikugawaGeneralHospital,Shizuoka,Japan,3DepartmentofInternalMedicine,OmaezakiMunicipalHospital,Shizuoka,Japan
Abstract
Background:Theanti-influenzavirusactivityofgreenteacatechinshasbeendemonstratedinexperimentalstudies,butclinicalevidencehasbeeninconclusive.School-agedchildrenplayanimportantroleintheinfectionandspreadofinfluenzaintheformofschool-basedoutbreaks.Preventinginfluenzainfectionamongstudentsisessentialforreducingthefrequencyofepidemicsandpandemics.Asanon-pharmaceuticalinterventionagainstinfection,garglingisalsocommonlyperformedinAsiancountriesbuthasnotyetbeenextensivelystudied.
MethodsandFindings:Arandomized,openlabel,2-groupparallelstudyof757highschoolstudents(15to17yearsofage)wasconductedfor90daysduringtheinfluenzaepidemicseasonfromDecember1st,2011toFebruary28th,2012,in6highschoolsinShizuokaPrefecture,Japan.Thegreenteagarglinggroupgargled3timesadaywithbottledgreentea,andthewatergarglinggroupdidthesamewithtapwater.Thewatergroupwasrestrictedfromgarglingwithgreentea.Theprimaryoutcomemeasurewastheincidenceoflaboratory-confirmedinfluenzausingimmunochromatographicassayforantigendetection.757participantswereenrolledand747participantscompletedthestudy(384inthegreenteagroupand363inthewatergroup).Multivariatelogisticregressionindicatednosignificantdifferenceintheincidenceoflaboratory-confirmedinfluenzabetweenthegreenteagroup(19participants;4.9%)andthewatergroup(25participants;6.9%)(adjustedOR,0.69;95%CI,0.37to1.28;P=0.24).Themainlimitationofthestudyistheadherencerateamonghighschoolstudentswaslowerthanexpected.
Conclusions:Amonghighschoolstudents,garglingwithgreenteathreetimesadaywasnotsignificantlymoreefficaciousthangarglingwithwaterforthepreventionofinfluenzainfection.Inordertoadequatelyassesstheeffectivenessofsuchgargling,additionallarge-scalerandomizedstudiesareneeded.TrialRegistration:ClinicalTrials.govNCT01225770
Citation:IdeK,YamadaH,MatsushitaK,ItoM,NojiriK,etal.(2014)EffectsofGreenTeaGarglingonthePreventionofInfluenzaInfectioninHighSchoolStudents:ARandomizedControlledStudy.PLoSONE9(5):e96373.doi:10.1371/journal.pone.0096373Editor:D.WilliamCameron,UniversityofOttawa,Canada
ReceivedDecember29,2013;AcceptedApril6,2014;PublishedMay16,2014
Copyright:ß2014Ideetal.Thisisanopen-accessarticledistributedunderthetermsoftheCreativeCommonsAttributionLicense,whichpermitsunrestricteduse,distribution,andreproductioninanymedium,providedtheoriginalauthorandsourcearecredited.
Funding:ThisworkwassupportedbyGrants-in-AidforScientificResearch(KAKENHI)GrantNumber23590887.Thefundershadnoroleinstudydesign,datacollectionandanalysis,decisiontopublish,orpreparationofthemanuscript.CompetingInterests:Theauthorshavedeclaredthatnocompetinginterestsexist.*E-mail:hyamada@u-shizuoka-ken.ac.jp
Introduction
Influenzaepidemicsareaperennialpublichealthproblemworldwide.Influenzacausesacuterespiratoryillness,andcanleadtoseverecomplicationswithmortalityrisk,suchaspneumoniaandencephalitis[1,2].Infectioninschoolsisespeciallyproblem-atic,ascloseinteractionamongstudentsallowsthevirustobeeasilytransmittedamongthem,andsubsequentlytotheirfamiliesandcommunities[3–6].Therefore,preventionisanessentialpublichealthmeasure.Themainstrategyforpreventinginfluenzainfectionisvaccination,butitsefficacyandeffectivenessdependonthestrainofthevirus[7],andithasthedrawbackoflimitedsupply[8,9].Neuraminidaseinhibitorsarealsousedforpreven-tion,butseveralreportshaveshownthattheyhavealimitedeffect,andthatviralresistancetoinhibitorssuchasoseltamivirhasbeen
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graduallyincreasing[10–14].Forthesereasons,avarietyofnon-pharmaceuticalpublichealthinterventionstoreducemorbidityhavebeensuggested,includingfacemasks,handhygiene,andgargling[15–17].InAsiancountries,andinJapanespecially,garglingisrecommendedandcommonlyperformed[18].Garglinghasnotbeenextensivelystudied,andtherehavebeenfewregisteredoutcomestudiesofitsrelationtoinfluenzainfection.However,onerandomizedtrialexaminingupperrespiratoryillnessfoundthatgarglingwithwaterreducedtherateofinfectionby36%comparedwithanon-garglingcontrolgroup[19].
Whileavarietyofnon-pharmaceuticalpublichealthinterven-tionshavebeensuggested,theydonotappeartohavehadasubstantialeffectoninfluenzainfectionrates.Infectionrateshaveremainedconsistentlyhigh,withthepeakpercentagesoftotal
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outpatientvisitstoU.S.healthcareprovidersforinfluenza-likeillnessbeing7.7%duringtheH1N1pandemicseasonin2009,and4.5%inthemostrecentlyreported2010-11season,accordingtotheOutpatientInfluenza-likeIllnessSurveillanceNetwork[20].Intermsofschool-relatedinfluenzainfections,atoneNewYorkCityhighschoolduringthe2009pandemicseason,therateofinfectionamongstudentswas3.5timeshigherthanamongtheschool’sstaff[21],indicatingpotentialinfectionroutesrelatedtohygieneandinteractionpatternsamongstudents.Thus,improvedpublichealthinterventionsperformedbystudentsmayhelptopreventepidemicsandpandemicsrelatedtoschool-basedoutbreaks.
Thepresentstudyfocusedonanovelnon-pharmaceuticalpublichealthinterventionagainstinfluenzaepidemicsinschools:garglingwithgreentea.Greenteaisoneofthemostwidelyconsumedbeveragesintheworld,anditschemicalcomponentssuchascatechinsandtheaninehaveavarietyofhealthbenefits[22,23].Experimentalstudieshaveshownthatgreenteacatechinshaveseveralanti-influenzavirusactivitiesinvitro.Regardinginfectivity,thehighly-bioactivecatechin(-)-epigallocatechingallatehasbeenreportedtoinhibitplaqueformation,adsorption,andhemmagglutinationbyinfluenzaAandBvirusesinMadin-Darbycaninekidneycells[24,25].Intermsofclinicalstudies,anumberofsmall-scalepreliminarystudiesontheuseofteacomponentsforinfluenzainfectionpreventionhavepreviouslybeenreported.Onestudysuggestedthatgarglingwithblackteaextractshadapreventiveeffectoninfluenzainfectionovera5-monthperiod;however,thediagnosticcriterionusedinthatstudywasbasedonhemagglutinineantibodytiterlevel,andthecontrolgroupdidnotgargle[26].Ourgrouphasalsocarriedoutclinicalstudiesontheuseofgreenteacomponentsforthepreventionofinfluenzainfection.Onewasasmallprospectivecohortstudyontheeffectivenessofgarglingwithteacatechinextractsamong124elderlynursinghomeresidents[27].Itfoundthatsuchgarglingsignificantlyreducedtherateofinfluenzainfection.Anotherstudywasarandomizedcontrolledtrialof200healthcareworkerswhichinvestigatedtheeffectthatcapsulesofcatechinsandtheaninefromgreenteahadonclinically-definedinfluenza[28].Thatstudyfoundthattherateofinfluenzainfectionwasapproximatelythreetimeslowerinthecatechin/theaninegroupthanintheplacebogroup.
Basedonthisbackground,weconductedarandomized,openlabel,2-groupparallelstudytoevaluatetheclinicalefficacyofgreenteagarglingonthepreventionofinfluenzainfectionamonghighschoolstudentsinJapan.
respiratory,renal,orhepaticdysfunctiondiagnosedbyamedicaldoctor.
Theparticipantscompletedaself-administeredquestionnairetoassessbaselinecharacteristicsincludingage,sex,bodymassindex(BMI),vaccinationforinfluenzavirus,useofpublictransportation,andtypeofschoolclub(sports-orculture-related).Greenteaconsumptionhabits(definedas$200mL/day)werealsorecorded.
RandomizationandInterventions
Eligibleparticipantswererandomizedbyacomputergeneratedpermutedblockrandomizedschema,andstratifiedaccordingtoschoolandclass,attheDataManagementCenterofShizuokaGeneralHospitalinJapan.Participantsinthegreenteagarglinggroupwereprovidedeachdaywithbottledgreentea(500mL)containingacatechinconcentrationof3760.2mg/dL,includingapproximately18%(-)-epigallocatechingallatemanufacturedbytheKakegawaTeaMerchantsAssociation.Participantsinthewatergarglinggroupwereaskedtogarglewithtapwater,andwereaskednottogarglewithgreenteaduringthestudy.Consumptionofgreenteaandotherteawasnotrestrictedforeithergroup.Theconcentrationofcatechinsintheteawasmeasuredbyhigh-performanceliquidchromatographybasedontheaverageconcentrationin10bottlesfromthesameproductionlot(September2011)usedforgarglinginthestudy.
Theparticipantswereaskedtogargleatleast3timesaday(afterarrivingatschool,afterlunch,andafterschool).Theywerealsoaskedtocompleteaquestionnaireeachdayconcerningtheoccurrenceofinfluenzainfection,preventivemeasures(handwashingandfacemasks),anyadverseevents,andtheirdailyadherencetothegarglingregimen.Thequestionnaireswerecollectedevery2weeksbythestudents’homeroomteachers,andsafetymonitoringwascarriedoutcarefullythroughoutthestudy.Datamanagementandsafetymonitoringwereassistedbythehighschools’viceprincipalsandheadteachers.
Allparticipantsandtheirguardiansgavewritteninformedconsentbeforeenteringthestudy.ThestudyprotocolwasapprovedbytheethicscommitteeoftheUniversityofShizuokaandwasconductedinaccordancewiththeDeclarationofHelsinki.
OutcomesandFollow-up
Theprimaryoutcomemeasurewastheincidenceoflaboratory-confirmedinfluenzainfectionwithviralantigendetectedbyimmunochromatographicassay.Thistestwasperformedwhenaninfluenza-likesymptomoccurred.Nasalswabsamplesforthisassaywerecollectedbymedicaldoctors.Thesensitivityofrapidinfluenzadiagnostictestsincludingtheimmunochromatographicassaywasapproximately64.4%(59.0–70.1%)fortypeAvirus,and52.2%(45.0–59.3%)fortypeBvirus[29].Twosecondaryoutcomemeasureswerealsoexamined.Thefirstwastheincidenceofclinicallydefinedinfluenzainfection,specifiedasfever($37.8uC)plusany2ofthefollowingadditionalsymptoms:cough,sorethroat,headache,ormyalgia[30].Theothersecondaryoutcomewasthetimeforwhichthepatientwasfreefromclinicallydefinedinfluenzainfection,i.e.,theperiodbetweenthestartoftheinterventionandthefirstdiagnosisofinfection.
MethodsDesignOverview
Arandomized,openlabel,2-groupparallelstudywasconduct-edtocomparetheefficacyofgreenteagarglingwithwatergarglingfor90daysduringtheinfluenzaepidemicseason,from1December2011to28February2012.TheprotocolforthisstudyandsupportingCONSORTchecklistareavailableassupportinginformation;seeChecklistS1andProtocolS1.
SettingandParticipants
Werecruitedatotalof2,838highschoolstudents(15to17yearsofage)whoattended6highschoolsintheKakegawaandOgasadistrictsofShizuokaPrefecture,Japan.Recruitmentwasperformedattheseschoolsbypostersandannouncementsatschoolassemblies.Participantswereexcludedaccordingtothefollowingcriteria:teaallergy(historyofasthma,skinrashes,andotherindications);historyofinfluenzainfectionwithintheprevious6months;andimmunediseaseorseverecardiac,
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StatisticalAnalysis
Basedonourpreviousstudies,weestimatedthatlaboratory-confirmedinfluenzainfection(primaryoutcome)wouldoccurin4%ofparticipantsinthegreenteagarglinggroupand10%ofthoseinthewatergarglinggroup.Thesamplesizewasestimatedas306foreachgroupatapowerlevelof0.80anda2-sidedalevel
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Figure1.Flowdiagramforstudy.doi:10.1371/journal.pone.0096373.g001
of0.05.Estimatinga15%dropoutrate,wesetthetotalsamplesizeat720.
Thefullanalysissetandperprotocolsetwereusedforallefficacyanalysis,andsafetyanalysiswasperformedforthefullanalysisset.Aninterimanalysiswasnotplanned.Thefullanalysissetwasdeterminedbyexcludingparticipantsfromtheintention-to-treatpopulationaccordingtothefollowingcriteria:nogarglingcarriedout,and/ornogarglingdatacollected,and/orwithdrawalfromthestudyandrefusaltohavedataincludedinthestudy.Inadditiontothesecriteria,theper-protocolsetwasdefinedaccordingtothefollowingcriteria:adherencerateofgarglingatorabove75%,andabsenceofgreenteagarglingwheninthewatergarglinggroup.
Achi-squaretestwasperformedforcategoricalcomparisonsofthedata.Differencesinthemeanvaluesofcontinuousmeasure-mentsweretestedbyStudent’st-testorMann-WhitneyUtest.Multivariatelogisticregressionanalysiswasusedtoprovideadjustedoddsratio(OR)estimatesanda95%confidenceinterval(CI)fortheassociationbetweengreenteagarglingandtheincidenceofinfluenzainfection.CumulativeincidenceratesweredeterminedbyKaplan-Meiermethod.TheCoxproportionalhazardsregressionmodelwasusedtoevaluatetheassociationbetweengreenteagarglingandthetimeforwhichtheparticipantswerefreefromclinicallydefinedinfluenzainfection,andwasadjustedforpotentialconfoundingvariablesafterconfirmationofthepopulationhazardassumption.Theparticipantsthatwerefreeofinfluenzainfectionforaperiodof90dayswerecensoredatthistime.BaselinecharacteristicswithP,0.20wereconsideredaspotentialconfoundingvariables.Theyweredefinedbymultivar-iatelogisticregressionanalysisandtransferredtotheCoxproportionalhazardmodel.Inthesemultivariateanalyses,vaccinationwasalsoconsideredanindependentvariablebecauseofitsmedicalimplications.
P,0.05wasconsideredstatisticallysignificant.AllstatisticalanalyseswereperformedusingSPSSforWindows,version21.0(IBMCorp.,Armonk,NY).
accordingtotheexclusioncriteria.Allofthe757participantswereenrolledandrandomlyassignedtoanintervention;387wereallocatedtothegreenteagarglinggroup,and370tothewatergarglinggroup.Betweenassignmentandintervention,3partici-pantsinthegreenteagroupand7inthewatergroupwithdrewfromthestudy.All10participantswhowithdrewdidsobecauseoftheirrefusaltogivetheirconsent.747participantscompletedthestudy(Figure1).Accordingtotheper-protocolsetcriteria,thegarglingadherenceratewas73.7%inthegreenteagroupand67.2%inthewatergroup.
BaselineCharacteristics
Thebaselinecharacteristicsoftheparticipantsinthefullanalysisset(n=747:greenteagroup,n=384;watergroup,n=363)andperprotocolset(n=527:greenteagroup,n=283;watergroup,n=244)areshowninTable1.Themeanageoftheparticipantswas16.1years,andtheagerangewas15to17years.Baselinecharacteristicswerewellbalanced,exceptfortypeofschoolclub(sport-orculture-related)(P=0.06inthefullanalysisset;P=0.17intheperprotocolset).Therefore,typeofschoolclubwasconsideredaconfoundingvariableinthemultivariateanalyses.Inaddition,theinfluenzavaccinationforuseinthe2011-12seasoncontainedthefollowingthreevaccineviruses:anA/California/7/2009(H1N1)pdm9-likevirus,anA/Perth/16/2009(H3N2)-likevirus,andaB/Brisbane/60/2008-likevirus,andthesubtypeofinfluenzaAviruscirculatinginJapanatthattimewasH3N2.ThevaccinationwasadministeredfromOctoberuntilmid-November,2011.
LaboratoryConfirmedInfluenzaInfectionandOtherOutcomes
Duringthestudy,laboratoryconfirmedinfluenzainfectionoccurredinatotalof44participants(5.9%)inthefullanalysisset,and36(6.8%)intheperprotocolsetparticipantswereinfected.Amongthe44confirmedparticipantsinthefullanalysisset,42hadtypeAantigen,and2hadtypeB.Clinicallydefinedinfluenzawasdiagnosedin113participants(14.9%)inthefullanalysissetand84(15.9%)intheperprotocolset.Noparticipantshadmorethanoneinfluenzainfectionduringtheobservationperiod.
Multivariatelogisticregressionanalysisshowedthattherewasnodifferencebetweenthegreenteaandwatergroupsinthe
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Results
Amongthe6highschoolsintheKakegawaandOgasadistricts,757of2,838studentsgavewritteninformedconsentandwereassessedforeligibility;noneofthestudentswereexcluded
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Table1.Baselinecharacteristicsofstudyparticipants.
FullanalysissetCharacteristicsAge,mean(SD)[range]Sex,No.(%)MenWomen
BMI,mean(SD)[range]
Vaccinationforinfluenzavirus,No.(%)Handwashing{{,No.(%)Facemasks{{,No.(%)Typeofclubs,No.(%)SportsCulture
Publictransportation,No.(%)
–GreenteadrinkinghabitI,No.(%)
Greenteagargling(n=384)16.1(0.7)[15–17]
Watergargling(n=363)16.2(0.7)[15–17]
Pvalue0.44{0.3310.23`0.6710.4310.7610.0610.2910.631224(58.3)160(41.7)
20.5(2.7)[15.4–36.7]139(36.2)312(81.3)52(13.5)
199(54.8)164(45.2)
20.6(2.4)[15.1–34.9]126(34.7)303(83.5)52(14.3)
253(65.9)131(34.1)181(47.1)270(70.3)Greenteagargling(n=283)16.1(0.7)[15–17]
214(58.9)149(41.1)157(43.3)261(71.9)Watergargling(n=244)16.1(0.7)[15–17]
PerprotocolsetCharacteristicsAge,mean(SD)[range]Sex,No.(%)MenWomen
BMI,mean(SD)[range]
Vaccinationforinfluenza,No.(%)Handwashing{{,No.(%)Facemask{{,No.(%)Typeofclub,No.(%)SportsCulture
Publictransportation,No.(%)
–GreenteadrinkinghabitI,No.(%)
Pvalue0.72{0.3210.37`0.5610.2910.4610.1710.9610.891163(57.6)120(42.4)
20.5(2.7)[15.4–36.7]109(38.5)243(85.9)41(14.5)
130(53.3)114(46.7)
20.6(2.4)[16.0–34.9]88(36.1)217(89.0)30(12.3)
179(63.3)104(36.7)134(47.3)208(73.5)
140(57.4)104(42.6)115(47.1)178(73.0)
Abbreviation:BMI,bodymassindex.{PvaluebasedonStudent’st-test.`PvaluebasedonMann-WhitneyUtest.1Pvaluebasedonchi-squaretest.{{Handwashingandfacemasksperformed4daysperweekormoreduringstudyperiod.I–Greenteadrinkinghabitdefinedasdrinkingover200mL(onecup)ofgreenteaperday.doi:10.1371/journal.pone.0096373.t001
incidenceoflaboratory-confirmedinfluenzainfection(Fullanalysisset:AdjustedOR,0.69;95%CI0.37to1.28;P=0.24.Perprotocolset:AdjustedOR,0.86;95%CI0.44to1.69;P=0.66)(Table2).Inaddition,therewasnosignificantdifferenceintheincidenceofclinicallydefinedinfluenzainthetwogroupsinthefullanalysisset(52participants(13.5%)inthegreenteagroup,and61participants(16.8%)inthewatergroup;AdjustedOR,0.75;95%CI0.50to1.13;P=0.17).Intheperprotocolset,theincidenceofclinicallydefinedinfluenzawaslowerinthegreenteagroup(39participants,13.8%)thaninthewatergroup(45participants,18.4%);however,thisdifferencewasnotsignificant(adjustedOR,0.69;95%CI0.43to1.11;P=0.13)(Table2).Kaplan-MeiercurvesareshowninFigure2.ThetimebeforediagnosisofclinicallydefinedinfluenzainfectionwasestimatedwiththeCoxproportionalhazardregressionmodel.Therewasnodifferencebetweenthetwogroups(Fullanalysisset:AdjustedHR,0.77;95%CI0.53to1.11;P=0.16.Perprotocolset:AdjustedHR,0.71;95%CI0.46to1.08;P=0.11)(Table3).
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Noadverseeventswereobservedintheparticipantsofeithergroupduringthestudy.
Discussion
ThisrandomizedclinicalstudywasconductedtocomparetheefficacyofgreenteagarglingwithwatergarglinginpreventinginfluenzainfectionamonghighschoolstudentsinJapan.Whileseveralpreviousexperimentalandclinicalstudieshadsuggestedthatteacomponents,especiallycatechins,wereeffectiveforpreventinginfluenzainfection,thisstudyofa90-daygreenteagarglinginterventionamonghighschoolstudentsdidnotshowasignificantdifferencecomparedwithwatergargling.
Itshouldbepointedout,however,thatthereareseverallimitationstothisstudy.Themainlimitationwasadherencerate.Inthesamplesizeestimation,theexpecteddropoutratewassetto15%,butinpracticethenon-adherencerateinthefullanalysissetwasmuchhigherat29%.Thereductionininfluenzainfectionwas
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Table2.Resultsofmultivariatelogisticregressionanalysisforinfluenzainfection.
Fullanalysisset
Laboratoryconfirmedinfluenza||VariableAllocationGreenteaWaterVaccinationClub(sportrelated)Perprotocolset
Laboratoryconfirmedinfluenza
VariableAllocationGreenteaWaterVaccinationClub(sportrelated)
||\"Clinicallydefinedinfluenza\"Pvalue
OR(95%CI)
Pvalue
OR(95%CI)
0.69(0.37to1.28)1[Reference]1.06(0.56to2.00)1.35(0.70to2.59)
0.240.75(0.50to1.13)1[Reference]
0.17
0.850.38
1.02(0.67to1.55)1.51(0.98to2.33)
0.940.07
Clinicallydefinedinfluenza
Pvalue
OR(95%CI)
Pvalue
OR(95%CI)
0.84(0.44to1.69)1[Reference]0.73(0.35to1.51)1.02(0.51to2.04)
0.660.69(0.43to1.11)1[Reference]
0.13
0.390.97
0.84(0.51to1.38)1.48(0.90to2.43)
0.490.13
Laboratoryconfirmedinfluenzawithviralantigendetectedbyimmunochromatographicassay.
Clinicallydefinedinfluenzainfectiondiagnosedasfever($37.8uC)plusany2offollowingsymptoms:cough,sorethroat,headache,ormyalgia.doi:10.1371/journal.pone.0096373.t002
greaterwhentheanalysiswererestrictedtotheperprotocolset;therefore,iftheadherenceratecouldbeimprovedinfuturestudies,itmaybepossibletodetectasignificantdifferencebetweenthewaterandgreenteagarglinggroups.Arelatedissueispracticalmethodsofensuringahighadherencerate.Inthisstudy,itwasdifficulttomaintainsucharateamongthehighschoolstudents.Itispossiblethattheyforgotorfoundtheprotocoltroublesome.Infuturestudies,theadherenceratemightbeimprovedbyinterventionssuchassendingreminderstoparticipants.Moreover,infuturestudiesitmaybepossibletodetectlaboratory-confirmedinfluenzainfectionmoreaccuratelybyusingmoresensitivediagnostictools.
Thenon-blindeddesignofthisstudymayalsobealimitation.Withthistypeofdesign,thereisthepotentialforplaceboeffects.However,itisdifficulttomakeaplacebogreenteabeveragebecauseoftheuniquetasteofgreentea.Thatbeingsaid,infuturestudiesitmaybeappropriatetoattempttousesuchabeverageinthecontrolgrouptoclearlyrevealtheeffectsofgreenteagargling.Doingsomayalsoimprovetheadherencerateofthegarglingcontrolgroup.Inthisstudy,theadherencerateofthewatergarglinggroup(67.2%)waslowerthanthatofthegreenteagarglinggroup(73.7%).Thismayberelatedtodifferencesinsustainingmotivationduetodifferencesintheparticipants’perceptionoftheeffectivenessoftheirtreatments.Therefore,theinterventionconditionsforthetwogroupsshouldbemadeassimilaraspossible.
Anadditionalpossiblelimitationisthelivingenvironmentoftheparticipants.Comparedwithourpreviousstudyofelderlynursing
Table3.ResultsofCoxproportionalhazardsregressionmodelforclinicallydefinedinfluenzainfection.
FullanalysissetVariableAllocationGreenteaWaterVaccinationClub(sportrelated)PerprotocolsetVariableAllocationGreenteaWaterVaccinationClub(sportrelated)
Abbreviation:HR,hazardratio;CI,confidenceinterval.doi:10.1371/journal.pone.0096373.t003
0.71(0.46to1.08)1[Reference]0.84(0.54to1.33)1.42(0.91to2.29)
0.460.120.11
HR(95%CI)
Pvalue
0.77(0.53to1.11)1[Reference]1.00(0.69to1.48)1.46(0.98to2.20)
0.970.060.16
HR(95%CI)
Pvalue
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Garglingfrequencymayalsohavebeenalimitation.Thefrequencywassetat3timesaday(afterarrivingatschool,afterlunch,andafterschool),basedonourpreviousstudyofgreenteagarglingamongelderlynursinghomeresidents.However,thisfrequencymaynothavebeenhighenoughtohaveaneffectonhighschoolstudents,whointeractwithothersatamuchhigherratethannursinghomeresidents,andthereforehavemanyopportunitiestobecomeinfected.Itispossiblethatmoredetaileddeterminationofthemosteffectivefrequencyanddurationofgarglingineachparticularpopulationandsitemayinfluencetherateofpreventionofinfection.
Baselinegreenteaconsumptionisalsoapotentiallimitationofthisstudy.Over70%oftheparticipantsinbothgroupswereregulargreenteadrinkers,consuming1cup(200mL)ormoreofthebeverageperday.Asourpreviousobservationalstudyofelementaryschoolstudentsindicatedthatconsumptionof3to5cups(600to1000mL)ofgreenteaperdaywasassociatedwithasignificantlyreducedincidenceofinfluenzainfection(adjustedORof0.62comparedwith,1cup/day)[31],thisbaselinegreenteaconsumptionratemayhaveinfluencedtheoutcomeofthepresentstudy.Infuturestudies,thequantityofgreenteaconsumeddailybyparticipantsshouldberecordedtocontrolfortheconfoundingeffectofgreenteaconsumption.
Inaddition,asahighrateofclinically-definedinfluenzainfectionwasfoundinthisstudy,itmaybefruitfulinfuturestudiestoexaminetheeffectofgarglingwithwaterorgreenteaincombinationwithotherpersonalhygienepracticesandhealthbehaviors,suchashandhygienewithethanol.Forexample,apreviousclinicalstudyonthecombinedeffectsofhandhygieneandfacemasksoninfectionpreventionfoundthat,whiletheuseoffacemasksalonewasnotefficaciousincomparisonwithanon-interventionalcontrol,thecombinationoffacemaskswithhandhygienesignificantlyreducedtheinfectionrate[16].
Insummary,thisstudyrevealsthat,inordertoadequatelyassesstheeffectsofgreenteagarglingonthepreventionofinfluenzainfection,additionallarge-scalerandomizedstudieswithsufficientlyhighadherenceratesandmaximallyeffectivegarglingmethodsareneeded.
SupportingInformation
ChecklistS1CONSORTchecklist.
(DOC)
ProtocolS1Studyprotocol.
(PDF)
Acknowledgments
Figure2.Kaplan-Meiercurvesforclinically-definedinfluenza.(A)Fullanalysisset,(B)Perprotocolset.doi:10.1371/journal.pone.0096373.g002
Wegratefullyacknowledgethestudentswhoparticipatedinthestudy,andtheteachingstaffsatKakegawaNishiHighSchool,KakegawaHigashiHighSchool,KakegawaTechnicalHighSchool,YokosukaHighSchool,OgasaHighSchool,andIkeshindenHighSchoolfortheirdedicatedcooperation.WethankPhilipHawkeoftheUniversityofShizuokaScientificEnglishProgramforhiscommentsontheEnglishinthemanuscript.
homeresidentswithameanageofover80years[27],theyoungparticipantsinthisstudyhadmanymoreopportunitiestobeinfectedwithinfluenzabecauseoftheircloseinteractionwitheachother.Thisobservationissupportedbystudiesshowingtheimpactofschoolclosuresonreducingtheseverityofinfluenzaepidemicsandpandemics,includingexcessdeathrate[3].Inaddition,thestudentswereinvolvedinavarietyofdailyactivitiesthatcarriedariskofinfection,suchastheuseofpublictransportation,aswellasmovementthroughothercrowdedplaces.
AuthorContributions
Conceivedanddesignedtheexperiments:KIHYKT.Performedtheexperiments:KIHYK.MatsushitaMIKNKT.Analyzedthedata:KIMIKNKTK.Matsumoto.Contributedreagents/materials/analysistools:HYYS.Wrotethepaper:KIHY.
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